Electrochemiluminescent monitoring of compounds

ABSTRACT

Detectable compounds comprising a chemically-transformable first compound covalently linked to an electrochemiluminescent compound are provided. Such compounds are useful in processes and kits that monitor the status of the first compound and derive information from such monitoring.

FIELD OF THE INVENTION

The present invention is directed generally to analytical biochemistry.More specifically, the present invention is useful for monitoringchemical transformations of detectable compounds having achemically-transformable first compound covalently linked to anelectrochemiluminescent compound.

BACKGROUND OF THE INVENTION

An ever-expanding field of applications exists for rapid, highlyspecific, sensitive, and accurate methods of detecting and quantifyingchemical, biochemical, and biological substances, including enzymes suchas may be found in biological samples. Because the amount of aparticular analyte of interest such as an enzyme in a typical biologicalsample is often quite small, analytical biochemists are engaged inongoing efforts to improve assay performance characteristics such assensitivity.

One approach to improving assay sensitivity has involved amplifying thesignal produced by a detectable label associated with the analyte ofinterest. In this regard, luminescent labels are of interest. Suchlabels are known which can be made to luminesce throughphotoluminescent, chemiluminescent, or electrochemiluminescenttechniques. "Photoluminescence" is the process whereby a materialluminesces subsequent to the absorption by that material of light(alternatively termed electromagnetic radiation or emr). Fluorescenceand phosphorescence are two different types of photoluminescence."Chemiluminescent" processes entail the creation of the luminescentspecies by a chemical reaction. "Electrochemiluminescence" is theprocess whereby a species luminesces upon the exposure of that speciesto electrochemical energy in an appropriate surrounding chemicalenvironment.

The signal in each of these three luminescent techniques is capable ofvery effective amplification (i.e., high gain) through the use of knowninstruments (e.g., a photomultiplier tube or pmt) which can respond onan individual photon by photon basis. However, the manner in which theluminescent species is generated differs greatly among and betweenphotoluminescent, chemiluminescent, and electrochemiluminescentprocesses. Moreover, these mechanistic differences account for thesubstantial advantages as an bioanalytical tool thatelectrochemiluminescence [hereinafter, sometimes "ECL"] enjoys visa visphotoluminescence and chemiluminescence. Some of the advantages possiblewith electrochemiluminescence include: (1) simpler, less expensiveinstrumentation; (2) stable, nonhazardous labels; and (3) increasedassay performance characteristics such as lower detection limits, highersignal to noise ratios, and lower background levels.

As stated above, in the context of bioanalytical chemistry measurementtechniques, electrochemiluminescence enjoys significant advantages overboth photoluminescence and chemiluminescence. Moreover, certainapplications of ECL have been developed and reported in the literature.U.S. Pat. Nos. 5,147,806; 5,068,808; 5,061,445; 5,296,191; 5,247,243;5,221,605; 5,238,808, and 5,310,687, the disclosures of which areincorporated by reference, detail certain methods, apparatuses, chemicalmoieties, inventions, and associated advantages of ECL.

Copending and commonly-assigned U.S. patent application Ser. No.08/368,429, filed Jan. 4, 1995, the disclosure of which is incorporatedby reference, details certain aspects of ECL in connection withbeta-lactam and beta-lactamase (neither of which is conjugated through acovalent linkage to an electrochemiluminescent compound).

None of the above-identified documents disclose nor suggest the presentinvention. Additionally, the practice of the invention offerssignificant advantages to the skilled bioanalytical chemist incomparison to the electrochemiluminescent techniques taught by thesedocuments. Accordingly, the invention meets the as-yet unmet needs ofskilled workers with respect to the achievement of improved assayperformance characteristics (e.g., signal output, detection limits,sensitivity, etc.) for the measured species and represents a patentableadvance in the field.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, processes, and kitsuseful for electrochemiluminescent monitoring of compounds. A criticalfeature of the invention which is common to these compounds, processes,and kits is detectable compounds comprising a chemically-transformablefirst compound covalently linked to an electrochemiluminescent compound.

In brief, these detectable compounds and their uses represent apatentable advance in the field of electrochemiluminescent measurementsbecause of their attributes. These attributes include the following:

1. They are electrochemiluminescent.;

2. They can be used to monitor chemically-transformable first compoundscovalently linked to electrochemiluminescent compounds.; and

3. The above-described monitoring can be extended to become an integralstep in performing assays for separate, nonconjugated compounds insample solutions (e.g., enzymes).

Applicants' present inventions are set forth immediately below in thefollowing nonexclusive, nonlimiting objects of the invention.

A first object of the invention is to provide electrochemiluminescentdetectable compounds comprising a chemically-transformable firstcompound covalently linked to an electrochemiluminescent compound.

A second object of the invention is to provide electrochemiluminescentprocesses for monitoring chemical transformations of the first compound.Consistent with this second object, assays are provided wherein thechemical transformation of the first compound is an integral step inperforming that assay.

A third object of the invention is to provide kits useful for practicingthe invention and for implementing the above-described first and secondobjects of the invention. Consistent with this third object, kits areprovided wherein at least one set of solutions containing the detectablecompounds is included.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a proposed ECL mechanism depicting reaction stepsassociated with the use of TPA as a nonconjugated reductant.

FIG. 2 shows a proposed ECL mechanism depicting reaction stepsassociated with the use of beta-lactam as a nonconjugated reductant.

FIG. 3(a-c) shows a proposed ECL mechanism depicting reaction stepsassociated with the use of a chemically-transformable first compound asa conjugated reductant.

FIG. 4 shows the synthesis of Ru-AMP.

FIG. 5 shows the mass spectrum of the ammonium hexafluorophosphate saltof Ru-AMP.

FIG. 6 shows the proton NMR spectrum of the ammonium hexafluorophosphatesalt of Ru-AMP.

FIG. 7 shows the synthesis of Ru-APA.

FIG. 8 shows the structures of 5 specific beta-lactams.

FIG. 9 shows the hydrolysis by NaOH or by beta-lactamase enzyme ofRu-AMP (left side) and of Ru-APA (right side).

FIG. 10 shows the comparison of measured ECL for a series of differentsamples.

FIG. 11 shows the comparison of measured ECL for a series of differentsamples.

FIG. 12 shows the effect of unhydrolyzed (closed circles) and hydrolyzed(open circles) Ru-AMP concentration on the measured ECL.

FIG. 13 shows the comparison of measured ECL for a series of differentsamples.

FIG. 14 shows the effect of unhydrolyzed (closed circles) and hydrolyzed(open circles) Ru-APA concentration on the measured ECL.

FIG. 15 shows the comparison of measured ECL for a series of differentsamples.

FIG. 16 shows a proposed ECL mechanism depicting reaction stepsassociated with the NADH-promoted ECL of Ru(bpy)₃ ⁺².

FIG. 17 shows the synthesis of Ru-NAD.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with detectable compounds comprising(A) a chemically-transformable first compound (B) covalently linked to(C) an electrochemiluminescent compound. The salient features of each ofthese three portions [(A), (B), and (C)] of the detectable compounds areindividually described below. Uses of the detectable compounds appear at(D) while specific examples of the present invention appear at (E).

A) The chemically-transformable first compounds

The terms "chemically-transformable first compound(s)" (hereinafter"CTFC") and "electrochemiluminescent compound(s)" (hereinafter "EC")each refer to the respective compound independent of certain minorvariations of that compound. The skilled worker will understand whichminor variation, if any, applies to any particular usage of either CTFCor EC by its context. The following explanations aid in theunderstanding of this context.

The term CTFC encompasses the following minor variations: (i) certainchanges in the formal redox state caused by reduction or oxidationreactions and certain chemical changes to the CTFC that do not destroythe covalent linkage between it and the EC (e.g., the ejection by theCTFC of H⁺¹); and (ii) certain chemical transformations (e.g., thehydrolysis of the CTFC) which alter the measurable luminescence of thedetectable compound in comparison to the measurable luminescence beforeany of such chemical transformations have occurred.

In comparing the measurable luminescence of the detectable compoundbefore and after such chemical transformation, several combinations arepossible as detailed in the chart below.

    __________________________________________________________________________    measurable                                                                    luminescence before                                                                     measurable luminescence after                                       __________________________________________________________________________    none      yes (an increase from zero)                                         yes       none (a decrease to zero)                                           yes       yes (an increase from nonzero)                                      yes       yes (a decrease from nonzero)                                       yes       yes (no change)      INOPERATIVE                                    none      none                 INOPERATIVE                                    __________________________________________________________________________

As depicted in this chart, the measurable luminescence of the detectablecompound is altered by the chemical transformation of the CTFC; i.e.,the measured luminescence before and after the chemical transformationdiffer from one another. However, there must be some measurableluminescence either before or after, or both before and after, any suchchemical transformation. Thus, the fifth and sixth entries in the abovechart do not represent compounds encompassed by the present inventionwhile the first four entries do represent compounds encompassed by thepresent invention.

FIG. 3(a-c) shows a proposed ECL mechanism depicting reaction stepsassociated with the use of a CTFC as a conjugated reductant that iscovalently linked to an EC. More particularly, the EC is exemplified bythe ruthenium (II) tris-bipyridyl cation (hereinafter "Ru(bpy)₃ ⁺² ")throughout FIG. 3(a-c). FIG. 3(a-c) illustrates contemplated minorvariations in these two compounds (i.e., in a CTFC and in an EC).

FIG. 3(a) depicts the postulated ECL mechanism for a detectable compoundcomprising a CTFC covalently linked to an EC. The chart below furtherexplains the depicted reactions.

    ______________________________________                                        Symbol       Definition                                                       ______________________________________                                        CTFC         electrochemically unchanged CTFC                                              (starting compound)                                              CTFC.sup..+1 radical, electrochemically oxidized CTFC                         CTFC.sup.. (-H.sup.+1)                                                                     radical, electrochemically neutral CTFC                                       formed by H.sup.+1 leaving CTFC.sup.+1 and able                               to act as a high-energy reductant in a                                        manner similar to TPA                                            CTFC(-H.sup.+1, -e.sup.-1)                                                                 electrochemically neutral, nonradical                                         CTFC formed by CTFC.sup.. (-H.sup.+1) intra-                                  molecularly donating an electron (e.sup.-1)                                   to the covalently linked EC                                      Ru(bpy).sub.3.sup.+2                                                                       nonexcited EC before electrochemical                                          oxidation                                                        Ru(bpy).sub.3.sup.+3                                                                       nonexcited EC after electrochemical                                           oxidation                                                        *Ru(bpy).sub.3.sup.+2                                                                      excited EC after being intramolecularly                                       reduced by the CTFC.sup.. (-H.sup.+1)                            Ru(bpy).sub.3.sup.+2                                                                       nonexcited, regenerated EC formed by                                          the emisson of light by excited EC                               hν        light emitted by the excited EC                                  ______________________________________                                    

FIG. 3(b) shows, relative to FIG. 3(a), all of the analogous reactionsto those of FIG. 3(a) with the exception that the symbol .sup.γ CTFC isconsistently used to represent the resulting chemically-transformed.sup.γ CTFC that is produced by the interaction between a CTFC and asecond compound (hereinafter "SC").

FIG. 3(c) shows the schematic depiction of the interaction between aCTFC with a SC to form the resulting .sup.γ CTFC.

The detectable compound depicted at FIG. 3 (a), (b), (c) represents acompound of the present invention that is able to produce measurableluminescence both before and after the chemical transformation of theCTFC. Thus, this compound exemplifies the third and fourth possiblecombinations of measurable luminescence contained in the previouslydiscussed chart. The depicted reactions are consistent with postulatedreaction mechanisms which culminate in measurable luminescence bothbefore and after the CTFC has been chemically transformed. For compoundsof the present invention falling within the first and second entries ofthe previously discussed chart (i.e., for those compound that onlyproduce measurable luminescence either before or (exclusively) after thechemical transformation of the CTFC), only the reaction mechanisms ofeither FIG. 3 (a) or (exclusively) FIG. 3 (b) is representative for anyparticular compound.

With regard to the minor variations of (i), both electrochemical redoxreactions and non electrochemical redox reactions are encompassed.Additionally, such changes are integral with and associated with thepostulated electrochemiluminescent mechanism including (a) steps leadingto the formation of a high-energy reductant as a form of the CTFC; and(b) steps leading to the actual luminescence by the EC.

With regard to the minor variations of (ii), chemical transformationsthat affect the intramolecular electron-donating ability of the CTFCwhen it acts as a high-energy reductant in electrochemiluminescencemechanisms are encompassed. The hydrolysis of a CTFC/substrate by eitherNaOH or an enzyme is an example of such a chemical transformation.

A critical feature of the CTFC is that it remains covalently linked tothe EC throughout all of the postulated reactions. Thus, it isunderstood that the previously discussed changes in and to the CTFCspecifically exclude changes which would destroy/break the covalentlylinkage of the CTFC to the EC. It is also understood that the changes inand to the CTFC alter the intramolecular electron-donating ability ofthe CTFC so that the measurable luminescence differs before and afterany such changes. The CTFC is chosen so that there is measurableluminescence either before or (exclusively) after, or both before andafter, any such changes.

Returning to the explanation of the term CTFC and the minor variationsencompassed therein, the scope of these variations is clear to theskilled worker.

The CTFC must be able to participate in the ECL mechanisms that causethe EC to luminesce. Specifically, the CTF C must function as ahigh-energy reductant capable of intramolecularly providing an electronto the EC so the EC is reduced into an excited (i.e., emissive) state.Suitable high-energy reductants for forming the excited state EC oftenhave an unpaired electron and are knows as radicals. FIG. 1 illustratesa proposed ECL mechanisms which uses TPA as a nonconjugated high-energyreductant. This mechanisms generates the actual high-energy reductant insitu subsequent to the initial electrochemical oxidation (triggering) ofthe TPA precursor. Suitable candidates for the CTFC of the presentinvention are within the knowledge of the skilled worker based on thedisclosure herein.

Applicants are not required to understand the theoretical underpinningswhich explain the observed behavior of the detectable compounds. Whilenot wishing to be bound by any particular scientific explanation forthese observed properties, applicants postulate the followingexplanations (I) and (II).

(I.) The ability of the covalently linked CTFC to act as a high-energyreductant by intramolecularly donating an electron to the EC variesaccording to whether that CTFC has or has not yet undergone a suitablechemical transformation. This variance can, depending upon theparticular CTF C involved, either increase or decrease the measuredluminescence in any one of the four previously discussed combinations.The chemical transformation in a CTFC resulting from the interactionwith a SC appears to cause this variance in intramolecularelectron-donating ability because of structural changes in the CTFCwhich might effect (i) the route through which the reducing electron hasto pass through; (ii) the ability of the reducing electron to begintraveling through any such route; or (iii) stereochemical/spatialorientation considerations.

(II.) The ability of the covalently linked CTFC to act as a high-energyreductant by intramolecularly donating an electron to the EC is greaterin comparison to the ability of that same (nonconjugated) CTFC tointermolecularly donate an electron to the EC. Correspondingly, themeasured luminescence for the detectable compounds of the presentinvention is greater in comparison with the measured luminescence ofelectrochemiluminescent compounds where the high energy reductant is notcovalently linked to the electrochemiluminescent compound.

Although nonconjugated high energy reductants are not the subject of thepresent invention; they nicely illustrate the importance of themechanistic differences. Certain electrochemiluminescent techniques,however, have focused on using such nonconjugated high energyreductants. FIGS. 1 and 2 illustrate proposed electrochemiluminescentmechanisms with such nonconjugated reductants. Specifically, FIG. 1depicts electrochemiluminescent reactions which use tri-n-propylamine(hereinafter "TPA") as such a reductant while FIG. 2 likewise depictsthese reactions with beta-lactam as the reductant. The postulatedelectrochemiluminescence mechanism shown in FIG. 1 using nonconjugatedTPA and Ru(bpy)₃ ⁺² has been previously reported in the literature. Thepostulated electrochemiluminescence mechanisms for beta-lactams(nonconjugated) shown in FIG. 2 [as noted previously, the use ofbeta-lactams as nonconjugated, high energy reductants inelectrochemiluminescence techniques is the subject of a copending andcommonly-assigned United States Patent Application] and for theconjugated high energy reductants of the present invention are derivedin part from and are thought to be consistent with the mechanisticexplanation for the TPA-induced electrochemiluminescence shown in FIG.1.

Applicants theorize the following explanation as to why, for example,beta-lactam as a nonconjugated reductant generates lesselectrochemiluminescence than beta-lactam as a conjugated reductant(i.e., as a CTFC). The nonconjugated beta-lactam must first diffusethrough solution to become sufficiently proximate to the EC and thenintermolecular donate an electron thereto. Moreover, during thisdiffusion process, the nonconjugated beta-lactam may react with anyavailable species (other than the EC) because it is a very reactive,radical species. In direct contrast, the CTFC does not have to diffusethrough the solution as a free species. The CTFC need onlyintramolecularly donate an electron to the covalently linked EC.

The above analysis teaches attributes of the CTFC sufficiently detailedto enable the skilled worker to practice the present invention. Toaugment the above teachings, applicants later provide examples usingparticularly-identified compounds as the CTFC. However, applicants'invention is not limited to any specific compounds; rather applicants'invention is limited only to suitable CTFC as taught by the foregoing.

(B) The covalent linkage

The covalent linkage comprises a linker group that covalently links oneof the chelating ligands of the EC to the CTFC. Thus, the near end ofthe linker group terminates with and extends into a covalent bondbetween an atom of the linker group and an atom of one of the chelatingligands of the EC while the far end of the linker group terminates withand extends into a covalent bond between an atom of the linker group andan atom of the CTFC.

This linker group must have the following attributes to ensure thatapplicants' detectable compounds are operative. As detailed below, theseattributes are divided into two main categories; namely, noninterferingand enhancing.

The noninterfering attributes are properties that the linker group musthave because otherwise their presence would interfere with theoperability of the invention. Specifically, the linking group during thecontemplated practice of the invention must not: (i) prohibit theelectrochemical reactions; (ii) prohibit the interactions between theCTFC and the SC; (iii) prohibit the overall electrochemiluminescencemechanism; and (iv) be itself destroyed by the necessary reactions ofthe invention. For example, a linker group containing anelectrochemically oxidizable species having a formal oxidation potentialclose to that of the central metal cation of the EC would not serve asan effective linking group.

The enhancing attributes of the linker group are those attributes thatspecifically relate to the ability of the CTFC to intramolecularlytransfer an electron to the central metal cation of the EC. Theseenhancing attributes include the length of the linking group and thenature of the bonds within such length. First, the length of theintervening linker group between the CTFC and the EC must (i) allow andpermit the appropriate intramolecular electron transfer to occur; and(ii) not prevent any necessary reaction from occurring due to steric orother considerations.

The term "intramolecular" transfer of an electron from the CTFC to theEC encompasses both transfer though bonds and through space. Such"intramolecular" transfers, however, are limited to transfers between adonating compound (i.e., the CTFC) and a corresponding receivingcompound (i.e., the EC) which are covalently linked to each otherthrough the linking group. The covalent linkage portion of thedetectable compounds must allow and permit at least one these two typesof intramolecular transfer.

For intramolecular transfer through bonds, the linker group must providesufficient delocalized, conductive electrons (e.g., conjugatedπ-systems) to enable the electron to travel through the bonds of thelinking group to than reach the central metal cation of the EC.

For intramolecular transfer through space, the linker group must enablethe CTFC to approach in relative close proximity the central metalcation of the EC. The linker group should be long enough andstereochemically flexible enough so that the CTFC attached to the farend of the linker group can swing back towards the metal cation and thenthe electron can intramolecularly transfer through the space thenseparating the CTFC and the EC. An additional limitation on theappropriate length of the linker group is that it should not be so longthat the frequency of the described swinging around effect (which effectis thought to be necessary for intramolecular transfer through space)significantly decreases. In the case of an excessively long linkergroup, the amount of luminescence produced would be lowered.

For example, a linking group that is not sufficiently long/flexible toenable intramolecular transfer through space and contains only saturatedbonds without any delocalized electrons (e.g., alkyl chains) would notbe an effective linker group.

There are several advantages that the linker group imparts to thedetectable compounds as compared to the electrochemiluminescentcompounds that are used with nonconjugated high-energy reductants. Thedetectable compounds of the present invention avoid the use of anydiffusion of free species through solutions. Possible advantages includea more rapid generation of the exited, luminescent-form of the EC andhigher signals associated with the more effective intramoleculartransfer of applicants' present invention as compared to theintermolecular transfer used with nonconjugated high-energy reductants.

This linkage also ensures that the ratio of the CTFC and the EC isone-to-one. This ratio is unlike that associated withelectrochemiluminescent techniques which use TPA nonconjugatedbeta-lactams as the high-energy reductant. Because of this ratio betweenthe two portion of the detectable compounds of the present invention,applicants are able to qualitatively and quantitatively monitor chemicaltransformations in the CTFC. Unlike known electrochemiluminescenttechniques, the compound monitored is simultaneously (i) covalentlylinked to the EC and (ii) capable of intramolecularly donating anelectron to the EC.

Suitable candidates to be tested as linker groups in the presentinvention are available to those of ordinary skill in the art. Inparticular, Vol. 136, Methods in Enzymology, K. Mosbach, Ed., pp. 3-30,Academic Press, NY (1987) discloses a series of "spacer molecules" forimmobilized active coenzymes, including NAD and ATP. The spacermolecules of this article, which article is fully incorporated byreference, are examples of such suitable candidates.

The above analysis, in connection with the disclosure herein, teachesattributes of the covalent linkage sufficiently detailed to enable theskilled worker to practice the present invention. Thus, the skilledworker can select appropriate candidates as linking groups anddetermine, by routine experimentation, those which do and do not work.To augment the above teachings, applicants later provide examples usingspecific detectable compounds having identified linker groups. However,applicants' invention is not limited to any such exemplified linkergroup. Rather, the present invention is limited only to covalentlinkages as taught herein to the skilled worker.

(C) The electrochemiluminescent compounds

The third and final portion of the detectable compounds are EC. Theseand their applications in certain contexts have been reported in theliterature. See, for example, the issued U.S. Patents previouslyincorporated by reference. The attributes and identities of such knownEC are known to skilled workers and need not be repeated in detail here.Thus, the term electrochemiluminescent compound is a term of art whosemetes and bounds are known to skilled workers. Nonlimiting, nonexclusiveexamples of particular detectable compounds (including the EC portion)and their uses are later provided.

The present invention, however, is not directed to EC in and ofthemselves nor is it directed to any of their known applications. Theinvention is directed to a novel and non-obvious use of EC; namely,their use in detectable compounds comprising a CTFC covalently linked toan EC. Accordingly, the skilled worker can practice the presentinvention in accordance with the disclosure herein in combination withthe existing knowledge of EC. Notwithstanding this, applicants provideguidelines for providing EC operative in the present invention.

The minor variations encompassed by the term CTFC discussed earlier at(A) apply in an analogous manner to those for the term EC and need notbe reexamined here. Thus, changes in formal redox state of the EC dueto, for example, electrochemical oxidation and intramolecular reductionas well as excited/nonexcited states are encompassed by the term EC andsuch changes represent acceptably differing forms of the EC.

The following formula (I.) depicts suitable electrochemiluminescentcompounds for use in the present invention:

    M(L.sup.1).sub.a (L.sup.2).sub.b (L.sup.3).sub.c (L.sup.4).sub.d (L.sup.5).sub.e (L.sup.6).sub.f                           (I.);

wherein

M is a central metal cation comprising ruthenium or osmium;

L¹ through L⁶ are each ligands of M, each of which may be monodentate orpolydentate, and each of which may be the same or different from eachother;

a through e are each 0 or 1;

provided that the ligands of M are of such number and composition thatthe compound can be induced to electrochemiluminescence; and

further provided that the total number of bonds provided by the ligandsto the central metal cation M equals the coordination number of M.

In the practice of the present invention, preferredelectrochemiluminescent compounds include those wherein the centralmetal cation is ruthenium Ru or osmium Os. A particularly preferredcompound is Ru(bpy)₃ ⁺².

Having established (i) that electrochemiluminescent compound is a termof art; (ii) guidelines for providing such EC; the term EC as usedherein is clear to skilled workers. Nonetheless, applicants lateramplify this teaching by providing nonlimiting, nonexclusive particularexamples which identify the EC.

(D) Uses of the detectable compounds

The identities, attributes, and theoretical basis of the detectablecompounds of the present invention have previously been detailed.Consequently, this section details the uses of such detectablecompounds.

The electrochemiluminescent processes that use the detectable compoundscan be viewed as being divided into two main categories; namely,monitoring and assaying.

The detectable compounds can be used to monitor chemical transformationsin the CTFC that alter the effective intramolecular donating ability ofthat CTFC. These monitoring processes are not primarily designed toqualitatively nor quantitatively identify the presence/amount of anyparticular SC. Rather, the monitoring processes are designed toqualitatively and/or quantitatively indicate the presence/extent ofchemical transformations in the CTFC without requiring identificationsdirected to which particular SC in the sample solution is responsiblefor any such chemical transformations.

By comparing (i) the measured luminescence of the detectable compoundafter the exposure of that detectable compound to sample solutionssuspected of containing at least one SC that is capable of interactingwith the CTFC and of effecting a chemical transformation in the CTFCwith (ii)the measured luminescence of the predetermined standard, theCTFC is effectively monitored. More specifically, the presence andextent of such chemical transformations in the CTFC can be monitored.The predetermined luminescence standard of the monitoring process isgenerated in the following manner.

The preparation of this calibration curve is illustrated for adetectable compound able to produce measurable luminescence before anychemical transformations in the CTF C. Known differing amounts of aparticular detectable compound are (in the purposeful absence of any SCcapable of interacting with the CTFC to cause a chemical transformation)prepared in a series of sample solutions. Each of these sample solutionsis caused to electrochemiluminescence upon exposure to electrochemicalenergy in the form of a positive voltage bias imposed on an electrode ofan electrochemiluminescent cell. The resulting experimentally measuredluminescence is recorded. The predetermined luminescence standard formonitoring techniques comprises a calibration curve havingexperimentally measured luminescence on a first axis and known amountsof the particular detectable compound on the second axis. By comparingthe experimentally measured luminescence of a solution containing aknown quantity of the detectable compound and also containing a samplesuspected of containing any second compounds with the correspondingluminescence value from the calibration curve, the CTFC is effectivelymonitored. Changes in the CTFC caused by interactions with any secondcompounds in the sample solution will result in measurable differences(deviations) from the calibration curve.

The monitoring processes can be used to screen suspected solutions foractivity against the CTFC. Specifically, a series of sample solutionscould be monitored with the detectable compounds. A positiveelectrochemiluminescence test result (i.e., a result that is eitherhigher or lower than the predetermined standard) for any particularsample solution is indicative of at least one SC in that particularsample solution. Accordingly, that solution would then be an appropriatecandidate for further detailed investigations.

The assaying processes are extensions of the monitoring processes inthat the assaying processes are designed to specifically test for thepresence and/or amount of a particular SC. As such, the assayingprocesses likewise are based on the chemical transformations in the CTFCwhich alter the effective intramolecular donating ability of the CTFC tothe EC.

By comparing (i) the measured luminescence of the detectable compoundafter the exposure of that detectable compound to a sample solutionsuspected of containing a particular SC that is capable of interactingwith the CTFC and of effecting a chemical transformation in the CTFCwith (ii)the measured luminescence of the predetermined standard, theparticular SC is effectively assayed. More specifically, the presenceand amount of the particular SC can be assayed. The predeterminedluminescence standard of the assaying process is generated in thefollowing manner.

Known amounts of a particular detectable compound are exposed to aseries of sample solutions each containing known differing amounts of aparticular SC that is capable of interacting with the CTFC of thedetectable compound in accordance with the present invention. Theexposure is effected under conditions favorable to and consistent withthe desired interactions. Subsequent to such interactions, each of thesesample solutions is caused to electrochemiluminescence and theexperimentally measured luminescence is recorded. The predeterminedluminescence standard for assaying processes comprises a calibrationcurve having experimentally measured luminescence on a first axis andknown amounts of the particular SC on the second axis.

For both monitoring and assaying processes, the experimentally measuredluminescence may be either greater than or less than the luminescencefor the applicable predetermined luminescence calibration curve. Inother words, the interaction between the CTFC and the at least onesecond compound may either increase or decrease the effectiveintramolecular electron donating ability of that CTFC (which wouldcorrespondingly increase or decrease the experimentally measuredluminescence).

Preferred applications of the detectable compounds are monitoring andassaying processes when the CTFC first compound comprises a substrateand the SC comprises an enzyme that is specific to that substrate.Particularly preferred substrates are beta-lactams. Such beta-lactamsare useful in assaying processes that test for the correspondingbeta-lactamase.

Another application of the detectable compounds of the present inventiontakes advantages of coupled, regenerative reaction mechanism thatinvolve the conversion of a separate, nonconjugated substrate insolution into a separate, nonconjugated product in solution via exposureto an appropriate enzyme and co-mediators. The interactions between theCTFC and the enzyme-catalyzed, co-mediated conversion of a substratespecies in solution to a product species in solution forms thetheoretical underpinnings for an assay that can be specific to thesubstrate in solution, the enzyme in solution, and/or the CTFC.

Another use of the detectable compounds of the invention are in kitsspecifically designed to implement the processes of the presentinvention. Accordingly two types of kits are provided. The monitoringkits each comprise a plurality of sample standard solutions eachcontaining known amounts of a particular detectable compound with thepurposeful absence of any SC. These monitoring kits can be used todetermine the predetermined luminescence standard calibration curve. Theassaying kits each comprise a plurality of sample solutions eachcontaining known amounts of a particular detectable compound in additionto a corresponding plurality of test solutions each containing knowndiffering amounts of a particular SC that is capable of interacting withthe detectable compound in the described manner.

(E) Examples

Notwithstanding the previous detailed description of the presentinvention, applicants below provide specific examples solely forpurposes of illustration and as an aid to understanding the invention.Particularly with respect to the protection to which the presentinvention is entitled to, these examples are both nonlimiting andnonexclusive. Accordingly, the scope of applicants' invention as setforth in the appended claims is to be determined in light of theteachings of the entire specification without incorporating in suchclaims the specific limitations of any particular example.

EXAMPLE 1

Preparation of Ru(bpy)₃ ⁺² -labeled beta-lactam antibiotics

(a) Preparation of Ru(bpy)₃ ⁺² -labeled ampicillin (Ru-AMP)

Ru(bpy)₃ ⁺² -NHS ester (15.1) mg in acetonitrile (250 μL) was mixed withampicillin (29.1 mg) in 0.2M sodium bicarbonate, pH 8.0 (250 μL) and thereaction was allowed to proceed at room temperature for 2 hours (FIG.4). Ru-AMP was purified using a Waters HPLC system (Milford, Mass.)equipped with a Progel™-TSJ CM-5PW column (7.5 cm×7.5 mm) (Supelco,Inc., Bellefonte, Pa.) using a 1.0 mL/minute, 15-minute linear gradientfrom 20-180 mM sodium phosphate, pH 7.0. Substrate was quantitatedspectrophotometrically by measuring the absorbance of the rutheniumcomplex (the molar extinction coefficient at 453 nm is 13,700 M⁻¹ cm⁻¹).Following formation of the ammonium hexafluorophosphate salt, thestructure and purity of Ru-AMP was confirmed by mass spectroscopy andproton NMR (FIGS. 5-6).

(b) Preparation of Ru(bpy)₂ ⁺² -labeled 6-aminopenicillanic acid(hereinafter "Ru-APA")

Ru(bpy)₃ ⁺² -NHS ester (15 mg) (IGEN, Inc., Gaithersburg, Md.) inacetonitrile (250 μL) was mixed with 6-aminopenicillanic acid (12.4 mg)in 0.2M sodium bicarbonate, pH 8.0 (350 μL) and the reaction was allowedto proceed at room temperature for 2 hours (FIG. 7). Ru-APA was purifiedwith a Waters HPLC system (Milford, Md.) equipped with a Progel™-TSKCM-5PW column (7.5 cm×7.5 mm) (Supelco, Inc., Bellefonte, Pa.) using a1.0 mL/minute, 20-minute linear gradient from 20-100 mM sodiumphosphate, pH 7.0. Substrate was quantitated spectrophotometrically bymeasuring the absorbance of the ruthenium complex (the molar extinctioncoefficient at 453 nm is 13,700 M⁻¹ cm⁻¹).

(c). Preparation of other Ru(bpy)₃ ⁺² -labeled beta-lactams

Other beta-lactams, such as 7-aminocephalosporanic acid, that have aprimary amine in their structures can also react with Ru(bpy)₃ ⁺² -NHSester to form similar conjugates as described above. The reaction andpurification conditions will be similar, potentially differing somewhatin ways solvable by one skilled in the art. FIG. 8 shows the structureof 5 specific beta-tactams.

EXAMPLE 2

ECL assay of Ru-AMP hydrolysis

Experiments were performed to compare the ECL properties of Ru-AMP(conjugated) with Ru(bpy)₃ ⁺² and ampicillin mixtures (nonconjugated).ECL properties were compared both before and after NaOH and enzymatichydrolysis (FIG. 9, left side).

Ru-AMP was found to be a very good substrate of beta-lactamase.Hydrolysis of Ru-AMP (33 μM) by beta-lactamase I from Bacillus cereus(0.3 nM) was monitored spectrophotometrically at 240 nm using a HitachiU3200 spectrophotometer (Danbury, Conn.) at 25.0° C. in 0.1M sodiumphosphate, pH 7.0. Half-time (t_(1/2)) analysis gave a k_(cat) /K_(m)for enzymatic hydrolysis of Ru-AMP of 3.9×10⁸ min⁻¹ M⁻¹.

The ECL properties of equimolar mixtures of Ru(bpy)₃ ⁺² and ampicillin(hydrolyzed or unhydrolyzed) were compared to the same concentration ofthe Ru-AMP conjugate (hydrolyzed or unhydrolyzed). In separateexperiments, ampicillin and Ru-AMP were hydrolyzed by either 250 mM NaOH(base hydrolysis) or 441 nM beta-lactam I from Bacillus cereus (enzymehydrolysis). For base hydrolysis, 50 μL of 5M NaOH were added to 1.0 mLsolutions of deionized water containing either 24.85 μM Ru-AMP or amixture of 25 μM ampicillin and 25 μM Ru(bpy)₃ ⁺². Following 30 minuteincubations, the solutions were neutralized with 50 μL of 5M HCl. Forthe unhydrolyzed counterpart experiments, 50 μL of H₂ O were added tosolutions of either 24.85 μM Ru-AMP or a mixture containing 25 μMampicillin and 25 μM Ru(bpy)₃ ⁺². Following 30 minute incubations, 50 μLof 5M NaCl was added to these solutions. The results shown in FIG. 10demonstrate: (1) that ampicillin hydrolysis by either NaOH orbeta-lactamase causes an increase in the ECL of the mixtures; and (2)that the increase in the ECL caused by the hydrolysis is dramaticallygreater when the light-emitting ruthenium complex is covalently linkedto ampicillin. With base hydrolysis, ECL increased 1.5-fold whenampicillin was hydrolyzed in a mixture of ampicillin and Ru(bpy)₃ ⁺²,while ECL increased 5.2-fold when Ru-AMP was hydrolyzed. Similar resultswere obtained in enzyme hydrolysis: ECL increased 2.1-fold whenampicillin was hydrolyzed in a mixture of ampicillin and Ru(bpy)₃ ⁺²,while ECL increased 9.8-fold upon hydrolysis of Ru-AMP. The dataestablishing these conclusions is found in FIG. 10 which shows theexperimentally measured electrochemiluminescence of (from left toright):

Ru(bpy)₃ ⁺² alone;

Ru(bpy)₃ ⁺² plus unhydrolyzed ampicillin;

Ru(bpy)₃ ⁺² plus NaOH-hydrolyzed ampicillin;

unhydrolyzed Ru-AMP;

NaOH-hydrolyzed Ru-AMP;

Ru(bpy)₃ ⁺² plus unhydrolyzed ampicillin;

Ru(bpy)₃ ⁺² plus beta-lactamase-hydrolyzed ampicillin;

unhydrolyzed Ru-AMP; and

beta-lactamase-hydrolyzed Ru-AMP.

This work was confirmed in a second experiment using enzyme hydrolysiswhich differed in that the incubation time with enzyme was lengthenedfrom 30 to 60 minutes (FIG. 11). Here, enzyme hydrolysis caused a2.5-fold increase in ECL when ampicillin and Ru(bpy)₃ ⁺² wereunconjugated and an 11.1-fold increase in ECL when the Ru-AMP conjugatewas hydrolyzed. The data establishing these conclusions is found in FIG.11 which shows the experimentally measured luminescence of (from left toright):

Ru(bpy)₃ ⁺² alone;

Ru(bpy)₃ ⁺² plus unhydrolyzed ampicillin;

Ru(bpy)₃ ² plus beta-lactamase-hydrolyzed ampicillin;

unhydrolyzed Ru-AMP; and

beta-lactamase-hydrolyzed Ru-AMP.

These results show that Ru(bpy)₃ ⁺² -conjugation caused intramoleculareffects that dramatically increase the experimentally measuredluminescence when the beta-lactam ring is hydrolyzed.

FIG. 12 shows that low concentrations of Ru-AMP can be detected byhydrolysis. The lower limit of detection was found to be 50 nM (464relative ECL counts for hydrolyzed Ru-AMP versus an average instrumentreading of -152 relative counts for unhydrolyzed Ru-AMP). This comparesfavorable to the lower limit for detection of (unconjugated) ampicillinhydrolysis which was 5000 nM.

EXAMPLE 3

ECL assay of Ru-APA hydrolysis

It was thought that Ru-APA might have different ECL properties (beforeand after hydrolysis) from those of Ru-AMP. The differences would be aconsequence of the structural differences between APA and AMP,especially the difference in distance between the beta-lactam ring andthe primary amino group used to conjugate Ru(bpy)₃ ⁺² -NHS ester (FIG.9, right side). In Ru-AMP, the beta-lactam ring is three bond lengthsfarther from the amino group than in Ru-APA. Specifically, hydrolysis ofRu-APA (or other beta-lactam conjugates) may be more or less sensitivelydetected by ECL than Ru-AMP hydrolysis.

The ECL properties of the Ru-APA conjugate were compared with those ofthe mixtures of unconjugated Ru(bpy)₃ ⁺² and 6-APA. ECL properties werecompared before and after NaOH and enzymatic hydrolysis. The data wasthen compared to the results of similar experiments with Ru-AMPdescribed in Example 2.

Ru-APA was found to be a very good substrate of beta-lactamase.Hydrolysis of Ru-APA (23 μM) by beta-lactamase I from Bacillus cereus(0.6 nM) was monitored spectrophotometrically at 240 nm using a HitachiU3200 spectrophotometer (Danbury, Conn.) at 25.0° C. in 0.1M sodiumphosphate, pH 7.0. Half-time (t₂) analysis gave a k_(cat) /k_(m) forenzymatic hydrolysis of Ru-APA of 9.8×10⁷ min⁻¹ M⁻¹. This rate indicatesthat the enzyme hydrolyzed Ru-APA with a 4-fold lower efficiency thanRu-AMP, but that Ru-APA hydrolysis by beta-lactamase is stillexceptionally efficient.

The ECL properties of equimolar mixtures of Ru(bpy)₃ ⁺² and APA(hydrolyzed or unhydrolyzed) were compared with the same concentrationof the Ru-APA conjugate (hydrolyzed or unhydrolyzed). In separateexperiments, 6-APA and Ru-APA were hydrolyzed by either 250 mM NaOH(base hydrolysis) or 347 nM beta-lactamase I from Bacillus cereus(enzyme hydrolysis).

For base hydrolysis, 50 μL of 5M NaOH were added to 1.0 mL solutions ofdeionized water containing either 23.0 μM Ru-APA or a mixture containing23.0 μM APA and 23.0 μM Ru(bpy)₃ ⁺². Following 60 minute incubations,the solutions were neutralized with 50 μL of 5M HCl. For unhydrolyzedcounterpart experiments, 50 μL of H₂ O were added to solutions of either23.0 μM Ru-APA or a mixture of 23.0 μM APA and 23.0 μM Ru(bpy)₃ ⁺².Following 60-minute incubations, 50 μL of 5M NaCl was added to thesesolutions. The results shown in FIG. 13 demonstrate: (1) that6-APA(conjugated or nonconjugated) hydrolysis by either NaOH orbeta-lactamase causes an increase in ECL; and (2) that the increase inECL caused by hydrolysis is dramatically greater when the light-emittingruthenium complex is covalently coupled to 6-APA. With base hydrolysis,ECL increased 1.9-fold when 6-APA (nonconjugated) in a mixture of 6-APAand Ru(bpy)₃ ⁺² was hydrolyzed, while ECL increased 13.2-fold whenRu-APA (conjugated) was hydrolyzed. Similarly with enzyme hydrolysis,ECL increased 1.4-fold when 6-APA (nonconjugated) in a mixture of 6-APAand Ru(bpy)₃ ⁺² was hydrolyzed, while ECL increased 31.8-fold whenRu-APA (conjugated) was hydrolyzed. The data establishing theseconclusions is found in FIG. 13 which shows the experimentally measuredluminescence of (from left to right):

Ru(bpy)₃ ⁺² alone;

Ru(bpy)₃ ⁺² plus unhydrolyzed 6-APA;

Ru(bpy)₃ ⁺² plus NaOH-hydrolyzed 6-APA;

unhydrolyzed Ru-APA;

NaOH-hydrolyzed Ru-APA;

Ru(bpy)₃ ⁺² plus unhydrolyzed 6-APA;

Ru(bpy)₃ ⁺² plus beta-lactamase-hydrolyzed 6-APA;

unhydrolyzed Ru-APA; and

beta-lactamase-hydrolyzed APA.

This work clearly demonstrates that conjugation of the 6-APA and theelectrochemiluminescent ruthenium complex result in intramoleculareffects that increase the electrochemiluminescence when the beta-lactamring is hydrolyzed. Moreover, comparison with the results described inExample 2 for the ampicillin conjugate show that hydrolysis of Ru-APAresults in a much greater electrochemiluminescence signal thanhydrolysis of Ru-AMP. Because the ruthenium atom is closer to thebeta-lactam ring in Ru-APA than in Ru-AMP, these results indicate thatthere may be a critical effect of the distance between the rutheniumcomplex and the beta-lactam ring. Other, as-yet untestedbeta-lactam-Ru(bpy)₃ ⁺² conjugates may give an even more dramatic changein the electrochemiluminescence upon beta-lactam hydrolysis.

FIG. 14 shows that the hydrolysis of very low concentrations of Ru-APAcan be detected by ECL. More specifically, FIG. 14 shows the effect ofunhydrolyzed (closed circles) and hydrolyzed (open circles) Ru-APAconcentration on the experimentally measured electrochemiluminescence.The lower limit of detection was found to be 50 nM (an instrumentreading of -33 relative ECL counts for hydrolyzed Ru-APA versus anaverage of -648 relative ECL counts for unhydrolyzed Ru-APA(conjugated).) This compares favorably to the lower limit for detectionof (unconjugated) APA hydrolysis which was 50 μM (in the presence of 10pM Ru(bpy)₃ ⁺²).

An experiment was performed to quantitate the advantage of conjugating abeta-lactam to the ECL label, Ru(bpy)₃ ⁺². The increase in ECL uponhydrolysis of 10 μM Ru-APA was compared to an ECL standard curve inwhich various concentrations of 6-APA (nonconjugated) were hydrolyzed inthe presence of 10 μM Ru(bpy)₃ ⁺². By extrapolation of the 6-APAstandard curve, the results (FIG. 15) demonstrates that the ECL changeupon hydrolysis of 10 μM Ru-APA (conjugated) is equivalent to the ECLchange in the hydrolysis of 1250 μM 6-APA (nonconjugated) in thepresence of 10 μM Ru(bpy)₃ ⁺². This demonstrates that conjugation ofRu(bpy)₃ ⁺² and 6-APA results in a 125-fold increase in the ECL changeseen during 6-APA hydrolysis. The data establishing these conclusions isfound at FIG. 15 which shows a comparison of electrochemiluminescenceeffects of Ru-APA (conjugated) to Ru(bpy)₃ ⁺² plus 6-APA (unconjugated).Triangles represent the electrochemiluminescence of 10 μM unhydrolyzed(open triangles) and hydrolyzed (closed triangles) Ru-APA. Circlesrepresent the electrochemiluminescence effects of unhydrolyzed (closedcircles) and hydrolyzed (open circles) 6-APA (0-1000 μM) in the presenceof 10 μM Ru(bpy)₃ ⁺². Extrapolation in FIG. 15 indicates theelectrochemiluminescence change upon hydrolysis of 10 μM Ru-APA isequivalent to the electrochemiluminescence change upon hydrolysis of1250 μM free 6-APA in the presence of 10 μM Ru(bpy)₃ ⁺².

EXAMPLE 4

Preparation of Ru(bpy)₃ ⁺² -labeled β-nicotinamide adenine cofactors

(a) Theory of Oxidoreductase Enzymes and Their Use in Assays

β-Nicotinamide adenine cofactors (such as NAD⁺, NADH, NADP⁺, NADPH) arewidely used in nature by oxidoreductase enzymes as oxidants orreductants during reduction or oxidation of metabolites. Such enzymesinclude many dehydrogenases (lactate dehydrogenase, alcoholdehydrogenase, glucose dehydrogenase, etc.). The oxidized forms of thesecofactors (NAD⁺ or NADP⁺) have little or no TPA-like effects in ECL.However, the reduced forms (NADH or NADPH) behave like TPA in promotingRu(bpy)₃ ⁺² electrochemiluminescence (1, 2). Consequently, ECL can beused to measure the enzyme-catalyzed formation or disappearance of thereduced forms of these cofactors. Hence, substrates (glucose, ethanol,etc.) of dehydrogenases can be detected by ECL since their chemicaltransformations by the appropriate enzyme stoichiometrically results inoxidation or reduction of nicotinamide adenine cofactors.

Reduced nicotinamide cofactors (NADH or NADPH) are not believed to bedestroyed during the ECL reactions as are TPA and beta-lactams, but areinstead converted to their oxidized forms (NAD⁺ or NADP⁺). This meansthat, in the presence of an appropriate dehydrogenase enzyme,nicotinamide adenine cofactors can be mused such that a single cofactormolecule that is covalently linked to an electrochemiluminescentcompound can participate in multiple ECL reactions (FIG. 16). Note alsoin FIG. 16 that the Ru(bpy)₃ ⁺² is also regenerated so that it ispossible for a single detectable compound comprising such a cofactorcovalently linked to an electrochemiluminescent compound can possiblyemit multiple photons one after another.

Nicotinamide adenine cofactors have advantages over presentelectrochemiluminescent techniques that use TPA. Specifically, thesecofactors (i) can participate in regenerative ECL reaction mechanisms;(ii) can be used to detect and quantitate dehydrogenases and theircorresponding substrates. One disadvantage is that the ECL signal (i.e.,the experimentally measured luminescence) is less in an ECL reactionwith NADH or NADPH than in an ECL reaction with TPA. This disadvantagecould be reduced or obviated by using a conjugate of derivatives orRu(bby)₃ ⁺² and the nicotinamide adenine cofactor. As shown in theExamples above, when Ru(bpy)₃ ⁺² is conjugated to achemically-transformable first compound which can act as a high energyreductant and intramolecularly donate an electron to the covalentlylinked electrochemiluminescent compound (such as a beta-lactam), the ECLsignal generated is much greater than when the CTFC is not conjugatedwith the EC. Similarly, a Ru(bpy)₃ ⁺² -nicotinamide adenine cofactor(reduced form) conjugate will also have more ECL than a nonconjugatedmixture of Ru(bpy)₃ ⁺² and the reduced cofactor. Similarly, thedifference in ECL signal between the reduced (NADH or NADPH) andoxidized forms (NAD⁺ NADP⁺) of the cofactors will be greater when thecofactors are covalently linked to the Ru(bpy)₃ ⁺² than when they arenot conjugated.

Conjugates of nicotinamide adenine cofactor derivatives are known andare enzymatically functional (3,4). One such cofactor derivative, N⁶-([6-aminohexyl]carbamoylmethyl) nicotinamide adenine dinucleotide, iscommercially available (Sigma Chem. Co., St. Louis, Mo.). The primaryamino group of this compound can be used to couple this compound to thesame Ru(bpy)₃ ⁺² -NHS ester described above (obtainable from IGEN, Inc.,Gaithersburg, Md.) by the same or similar method (FIG. 17) (3,4). Othersimilar coupling methods will also work. The conjugate (Ru-NAD) can bepurified by HPLC in a similar manner as described for purification ofRu-AMP and Ru-APA. The four references noted above are (1) Downey, T. M.& Nieman, T. A. (1992) Anal. Chem. 64, 261-268; (2) Martin, A. F. &Nieman, T. A. (1993) Anal. Chem. Acta. 281, 475-481; (3) Mansson, M.-O.,Larsson, P.-O, & Mosbach, K. (1982) Methods Enzym. 89, 457-468; and (4)Persson, M., Mansson, M. O., Bulow, L., Mosbach, K. (1991)Bio/Technology 9, 280-284. Each of these four references is incorporatedby reference. FIG. 17 shows the preparation of Ru-NAD.

The oxidized form of Ru-NAD (Ru-NAD⁺) can be used in enzyme assays in anECL instrument to detect and quantitate a dehydrogenase enzyme or asubstrate of a dehydrogenase (or some compound that gives rise toeither). The assays will be performed according to conventionalprotocols (duration, temperature, pH, buffer, salt, substrate and enzymeconcentrations, etc.) except that NAD⁺ normally included will beexcluded and Ru-NAD⁺ will be used in place. The concentration of Ru-NAD⁺may be lower or higher than the conventional assays owing to differencesin substrate specificity, solubility, cost, or other factors. Followingthe incubation, the mixture will be analyzed in an ECL instrument (IGEN,Inc., Gaithersburg, Md.). No additional Ru(bpy)₃ ⁺² will be added.Reduction of Ru-NAD⁺ will be recognized by an increase in ECL signalover background and will indicate the presence of the relevantdehydrogenase and substrate.

Similarly, oxidation of the reduced form of Ru-NAD (Ru-NADH) can bedetected by ECL. Again, conditions, and the presence of relevant enzymeand enzyme substrate will be considered and will be derived from knownconditions for assays involving nonconjugated NADH. NADH will be omittedfrom the assay and Ru-NADH (at an appropriate concentration that may notbe the conventional concentration) will be included. Followingincubation, the mixture will be analyzed with an ECL instrument. Anydecrease in ECL from the initial Ru-NADH signal will indicate that someRu-NADH has been oxidized and will be evidence of the presence of therelevant enzyme or substrate.

Preparation of Ruthenium-Labelled N⁶ -aminohexyl-(carbamoylmethyl)-NAD⁺

To a solution containing 6.6 mg N⁶ [6-aminohexyl-(carbamoylmethyl)-NAD⁺(Li⁺ salt, Sigma Chem. Co., St. Louis, Mo.) in 0.4 mL of a 1:1 mixtureof acetonitrile and NaHCO₃ (0.2 M, pH 8.6) was added an NHS ester ofRu(bpy)₃ ²⁺ (IGEN, Inc., Gaithersburg, Md.) in 0.2 mL of a 1:1 mixtureof acetonitrile and NaHCO₃ (0.2M, pH 8.6). The reaction mixture was runovernight at room temperature. The following morning, the reaction wasstopped, the solvent removed, and the compound was purified by sizeexclusion chromatography (BioRad Bio-Gel P-2, BioRad Laboratories,Richmond, Calif.). Proton NMR showed the compound to be correct, but notcompletely pure. The compound (Ru-NAD) was repurified on a column ofSp-Sephadex (Pharmacia, Uppsala, Sweden), eluting with changes ofincreasing concentrations of trifluoroacetic acid (0, 0.05, 0.2, 0.3M).NMR showed to compound to be pure Ru-NAD.

(c) Ru-NAD as an Enzyme Cofactor

To determine whether Ru-NAD was functional as an enzyme cofactor, areaction involving oxidation of D-glucose-6-phosphate byglucose-6-phosphate dehydrogenase was tested. The reaction was monitoredspectrophotometrically at 340 nm. This wavelength is commonly used toobserve the interconversion of NAD⁺ and NADH. A mixture of 63 μM Ru-NAD,400 μM glucose-6-phosphate, and 22 nM enzyme in 55 mM Tris buffer, pH7.8 containing 33 mM MgCl₂ was incubated at 30° C. in a cuvette.Continuous absorbance readings showed that absorbance increased overapproximately 40 minutes in a fashion characteristic of enzymaticreduction of NAD⁺. This indicated that Ru-NAD was indeed accepted as afunctional cofactor by glucose-6-phosphate dehydrogenase.

(d) Effect of Enzymatic Reduction on the ECL of Ru-NAD

Ru-NAD was found to be accepted as a cofactor by the dehydrogenase,glucose-6-phosphate dehydrogenase. An experiment was performed involvingoxidation of glucose-6-phosphate by this enzyme with concurrentreduction of Ru-NAD. Here, ECL measurements were made to determine if;(1) the ECL-inducing effects of NADH (but not NAD⁺) are also present inRu-NADH (but not Ru-NAD) and (2) if conjugation of Ru(bpy)₃ ²⁺ with NADHcauses an increase in ECL measurement sensitivity as compared to the ECLof a mixture of unconjugated Ru(bpy)₃ ²⁺ and NADH. The results are shownbelow (all solutions contain the substrate, glucose-6-phosphate,solutions not containing Ru-NAD contained 1.0 μM Ru(bpy)₃ ⁺²);

    ______________________________________                                        Sample             ECL counts                                                 ______________________________________                                        21 μM NAD.sup.+ 45,500                                                     21 μM NAD.sup.+ 45,200yme                                                  21 μM NADH      47,900                                                     21 μM NADH + enzyme                                                                           40,800                                                     21 μM Ru-NAD    71,700                                                     21 μM Ru-NAD + enzyme                                                                         132,000                                                    ______________________________________                                    

These results show that addition of enzyme to Ru-NAD increases the ECLsignal. Also the results show that, at unconjugated NAD concentrationstoo low for ECL effects to be seen, Ru-NAD clearly gives a large amountof ECL when enzyme is added. In conclusion, Ru-NAD behaves in the sameway as free Ru(bpy)₃ ⁺² plus free NAD⁺ in an ECL instrument (enzymeaddition causes an increase in ECL), but Ru-NAD is much more sensitivelydetected. This indicated that low concentrations of dehydrogenases ortheir substrates can be sensitively detected by ECL of Ru-NAD⁺ reductionor Ru-NADH oxidation.

The scope of the patent protection which the present invention isentitled to is not limited by the preceding text. Rather, the presentinvention is defined by the claims appended hereto and all embodimentsfalling thereunder.

We claim:
 1. A detectable electrochemiluminescent compound useful formonitoring changes in compounds, comprising:(a) achemically-transformable first compound covalently linked to anelectrochemiluminescent compound; wherein(I) the first compound is asubstrate capable of (i) being chemically transformed upon aninteraction with at least one second compound which is an enzymespecific to said substrate; (ii) intramolecularly donating an electronto the electrochemiluminescent compound to cause theelectrochemiluminescent compound to subsequently luminesce; and (iii)varying in its ability to effect such intramolecular donation of anelectron before and after any interaction; and (II) upon the exposure ofthe detectable compound to electrochemical energy the first compoundintramolecularly donates an electron to the electrochemiluminescentcompound to cause the electrochemiluminescent compound to luminesce. 2.The electrochemiluminescent compound of claim 1, wherein:the substrateis a beta-lactam and the enzyme is a beta-lactamase.
 3. Theelectrochemiluminescent compound of claim 1, wherein:the beta-lactam isselected from the group consisting of penicillin G, ampicillin,moxalactam, amoxicillin, cefoxitin, 6-aminopencillanic acid,7-aminocephalosporanic acid, cephalosporin C, cefaclor, and cefuroxime.4. The electrochemiluminescent compound of claim 1, wherein thedetectable compound has the structure: ##STR1##
 5. Theelectrochemiluminescent compound of claim 1, wherein the detectablecompound has the structure: ##STR2##
 6. The electrochemiluminescentcompound of claim 1, wherein the detectable compound has the structure:##STR3## and wherein R is ribose and P is phosphate.
 7. Theelectrochemiluminescent compound of claim 1, wherein:(a) the firstcompound is a comediator of an enzyme catalyzed conversion of anonconjugated substrate to a nonconjugated product.
 8. Anelectrochemiluminescent process for monitoring chemical transformationsof compounds, comprising:(a) contacting a detectable compound as definedin claim 1 to a sample solution suspected of containing at least onesecond compound; (b) exposing the detectable compound to electrochemicalenergy to cause electrochemiluminescence; (c) measuring the luminescenceemitted by the detectable compound; and (d) monitoring the presence ofany such chemical transformations of the first compound by comparing themeasured luminescence with a predetermined standard.
 9. The process ofclaim 8, wherein the monitoring step further includes quantitativelycalculating the extent to which the first compound has been chemicallymodified.
 10. The process of claim 8, wherein the monitoring step,independent of the actual identification of any second compound, furtherdetermines whether there are any second compounds in the sample solutioncapable of effecting such chemical transformations in the first compoundand wherein the process is repeated with a series of sample solutions ina screening fashion that is capable of selecting, based on the measuredluminescence, any particular sample solution for further investigation.11. The process of claim 8, wherein the determining step is part of aprocess for performing an assay for at least one particular secondcompound whose presence or absence is determined from the measuredluminescence of step (d) and wherein a particular detectable compoundcorresponding to and capable of being chemically transformed by exposureto the particular second compound is used in the process.
 12. Theprocess of claim 11, wherein the determining step further includesquantitatively calculating the amount of the particular second compound.13. The process of claims 11, wherein the first compound is a substrateand the second compound is an enzyme both specific to the first compoundand capable of catalyzing the chemical transformation of the firstcompound upon the interaction therewith.
 14. The process of claim 13,wherein the substrate is a beta-lactam and the enzyme is abeta-lactamase.
 15. The process of claim 14, wherein the beta-lactam isselected from the group consisting of 6-aminopencillanic acid,ampicillin, 7-aminocephalosporanic acid, penicillin G, amoxicillin,moxalactam, cefotin, cephalosporin C, cefaclor, and cefuroxime.
 16. Theprocess of claim 15, wherein the detectable compound has the formula:##STR4##
 17. The process of claim 15, wherein the detectable compoundhas the formula: ##STR5##
 18. The process of claim 12, wherein thedetectable compound formula: ##STR6## and wherein R is ribose and P isphosphate.
 19. A kit useful for preparing a predetermined luminescencestandard, comprising:(a) a plurality of test sample solutions eachcontaining differing known amounts of a particular detectable compoundas defined in claim 1; provided that each of the solutions does notcontain any second compounds; and provided that each of the test samplesolutions is capable of being induced to electrochemiluminescence atexperimentally measured levels that are sufficiently distinct from oneanother so that a luminescence calibration curve can be constructed fromsuch levels.
 20. A kit useful for preparing a predetermined luminescencestandard, comprising:(a) a plurality of first test solutions eachcontaining known amounts of a particular detectable compound as definedin claim 1; provided that each of the first solutions does not containany second compounds; (b) a plurality of second test solutionsrespectively matching in total number the number of first test samplesolutions, each of the second test solutions containing differingamounts of a particular second compound corresponding to and capable ofeffecting a chemical transformation in the particular detectablecompound; provided that the set of first test solutions and the set ofsecond test solutions are ready to be mixed, under conditions permittingany such chemical transformations to occur, pairwise with each other toform a set of third test solutions; and provided that each of the thirdtest solutions is capable of being induced to electrochemiluminescenceat experimentally measured levels that are sufficiently distinct fromone another so that a luminescence calibration curve can be constructedfrom such levels.